Electrochemical cell

ABSTRACT

A biosensor for use in determining a concentration of a component in an aqueous liquid sample is provided including: an electrochemical cell having a first electrically resistive substrate having a thin layer of electrically conductive material, a second electrically resistive substrate having a thin layer of electrically conductive material, the substrates being disposed with the electrically conductive materials facing each other and being separated by a sheet including an aperture, the wall of which aperture defines a cell wall and a sample introduction aperture whereby the aqueous liquid sample may be introduced into the cell; and a measuring circuit.

RELATED APPLICATIONS

This application is a continuation of copending U.S. application Ser.No. 09/709,968, filed Nov. 10, 2000, which is a continuation ofcopending U.S. application Ser. No. 09/314,251, filed May 19, 1999,which issued as U.S. Pat. No. 6,174,420 on Jan. 16, 2001, which is acontinuation of copending U.S. application Ser. No. 09/068,828, filed onMay 15, 1998, and of copending U.S. application Ser. No. 08/852,804,filed on May 7, 1997, which issued as U.S. Pat. No. 5,942,102 on Aug.24, 1999, the contents of which are incorporated herein by reference intheir entirety.

FIELD OF THE INVENTION

This invention relates to an electrochemical cell for determining theconcentration of an analyte in a carrier.

BACKGROUND OF THE INVENTION

The invention herein described is an improvement in or modification ofthe invention described in our co-pending U.S. application Ser. No.08/981,385, entitled ELECTROCHEMICAL CELL, filed on Dec. 18, 1997, thecontents of which are incorporated herein by reference in its entirety.

The invention will herein be described with particular reference to abiosensor adapted to measure the concentration of glucose in blood, butit will be understood not to be limited to that particular use and isapplicable to other analytic determinations.

It is known to measure the concentration of a component to be analysedin an aqueous liquid sample by placing the sample into a reaction zonein an electrochemical cell comprising two electrodes having an impedancewhich renders them suitable for amperometric measurement. The componentto be analysed is allowed to react directly or indirectly with a redoxreagent whereby to form an oxidisable (or reducible) substance in anamount corresponding to the concentration of the component to beanalysed. The quantity of the oxidisable (or reducible) substancepresent is then estimated electrochemically. Generally this methodrequires sufficient separation of the electrodes so that electrolysisproducts at one electrode cannot reach the other electrode and interferewith the processes at the other electrode during the period ofmeasurement.

In our co-pending application we described a novel method fordetermining the concentration of the reduced (or oxidised) form of aredox species in an electrochemical cell of the kind comprising aworking electrode and a counter (or counter/reference) electrode spacedfrom the working electrode by a predetermined distance. The methodinvolves applying an electric potential difference between theelectrodes and selecting the potential of the working electrode suchthat the rate of electro-oxidation of the reduced form of the species(or of electro-reduction of the oxidised form) is diffusion controlled.The spacing between the working electrode and the counter electrode isselected so that reaction products from the counter electrode arrive atthe working electrode. By determining the current as a function of timeafter application of the potential and prior to achievement of a steadystate current and then estimating the magnitude of the steady statecurrent, the method previously described allows the diffusioncoefficient and/or the concentration of the reduced (or oxidised) formof the species to be estimated.

Our co-pending application exemplifies this method with reference to useof a “thin layer electrochemical cell” employing a GOD/Ferrocyanidesystem. As herein used, the term “thin layer electrochemical cell”refers to a cell having closely spaced electrodes such that reactionproduct from the counter electrode arrives at the working electrode. Inpractice, the separation of electrodes in such a cell for measuringglucose in blood will be less than 500 microns, and preferably less than200 microns.

The chemistry used in the exemplified electrochemical cell is asfollows:glucose+GOD→gluconic acid+GOD*   reaction 1GOD*+2ferricyanide→>GOD+2ferrocyanide reaction   2where GOD is the enzyme glucose oxidase, and GOD* is the ‘activated’enzyme. Ferricyanide ([Fe(CN)₆·³ is the ‘mediator’ which returns theGOD* to its catalytic state. GOD, an enzyme catalyst, is not consumedduring the reaction so long as excess mediator is present. Ferrocyanide([Fe(CN)₆]⁴⁻) is the product of the total reaction. Ideally there isinitially no ferrocyanide, although in practice there is often a smallquantity. After reaction is complete the concentration of ferrocyanide(measured electrochemically) indicates the initial concentration ofglucose. The total reaction is the sum of reactions 1 and 2:

“Glucose” refers specifically to P-D-glucose.

The prior art suffers from a number of disadvantages. Firstly, samplesize required is greater than desirable. It would be generallypreferable to be able to make measurements on samples of reduced volumesince this in turn enables use of less invasive methods to obtainsamples.

Secondly, it would be generally desirable to improve the accuracy of &measurement and to eliminate or reduce variations due, for example, tocell asymmetry or other factors introduced during mass production ofmicrocells. Likewise, it would be desirable to reduce electrode “edge”effects.

Thirdly, since the cells are disposable after use, it is desirable thatthey be capable of mass production at relatively low cost.

SUMMARY OF THE INVENTION

In a first embodiment of the present invention, a biosensor for use indetermining a concentration of a component in an aqueous liquid sampleis provided, the biosensor including: (a) an electrochemical cell, theelectrochemical cell including a first electrically resistive substratehaving a first thin layer of a first electrically conductive material ona first face, a second electrically resistive substrate having a secondthin layer of a second electrically conductive material on a secondface, the substrates being disposed with the first electricallyconductive material facing the second electrically conductive materialand being separated by a sheet including an aperture, the wall of whichaperture cooperates with the electrically conductive materials to definea cell wall, and wherein the aperture defines a working electrode areain the cell, the cell further including a sample introduction aperturewhereby the aqueous liquid sample may be introduced into the cell; and(b) a measuring circuit.

In one aspect of the first embodiment, the electrochemical cell furtherincludes a socket region having a first contact area in electricalcommunication with the first thin layer of the first electricallyconductive material and a second contact area in electricalcommunication with the second thin layer of the second electricallyconductive material, whereby the electrochemical cell may beelectrically connected with the measuring circuit.

In another aspect of the first embodiment, the measuring circuitincludes a tongue plug.

In a further aspect of the first embodiment, at least one of the firstelectrically conductive material and the second electrically conductivematerial includes a metal.

The metal may further include a sputter coated metal.

In still other aspects of the first embodiment, the aqueous liquidsample includes blood, and the component includes glucose.

In yet another aspect of the first embodiment, the measuring circuitincludes an automated instrument for detecting an electrical signal fromthe electrochemical cell and relating the electrical signal to theconcentration of the component in the aqueous liquid sample.

In a further aspect of the first embodiment, the electrochemical cellincludes a substantially flat strip having a thickness, the strip havingat least two lateral edges, and wherein the sample introduction apertureincludes a notch through the entire thickness of the strip in at leastone of the lateral edges thereof.

In a second embodiment of the present invention, a biosensor for use ine determining a concentration of a component in an aqueous liquid sampleis provided, the biosensor including: (a) a thin layer electrochemicalcell, the cell including: (i) an electrically resistive sheet includingan aperture wherein the aperture defines a working electrode area in thecell; (ii) a first electrode layer covering the aperture on a first sideof the sheet; (iii) a second electrode layer covering the aperture on asecond side of the sheet; and (iv) a passage for admission into theaperture of the aqueous liquid sample; and (b) a measuring circuit.

In one aspect of the second embodiment, the electrochemical cell furtherincludes a socket region having a first contact area in electricalcommunication with the first electrode layer and a second contact areain electrical communication with the second electrode layer, whereby theelectrochemical cell may be electrically connected with the measuringcircuit.

In another aspect of the second embodiment, the measuring circuitincludes a tongue plug.

In still other aspects of the second embodiment, the aqueous liquidsample includes blood, and the component includes glucose.

In a further aspect of the second embodiment, the measuring circuitincludes an automated instrument for detecting an electrical signal fromthe electrochemical cell and relating the electrical signal to theconcentration of the component in the aqueous liquid sample.

In yet another aspect of the second embodiment, the cell includes asubstantially flat strip having a thickness, the strip having at leasttwo lateral edges, and wherein the passage for admission into theaperture includes a notch through the entire thickness of the strip inat least one of the lateral edges thereof.

In a third embodiment of the present invention, an apparatus fordetermining a concentration of a reduced form or an oxidized form of aredox species in a liquid sample is provided, the apparatus including:(a) a hollow electrochemical cell having a working electrode and acounter or counter/reference electrode wherein the working electrode isspaced from the counter or counter/reference electrode by less than 500μm; (b) means for applying an electric potential difference between theelectrodes; and (c) means for electrochemically determining theconcentration of the reduced form or the oxidized form of the redoxspecies in the liquid sample.

In one aspect of the third embodiment, means for electrochemicallydetermining the concentration of the reduced form or the oxidized formof the redox species includes: (i) means for determining a change incurrent with time after application of the electric potential differenceand prior to achievement of a steady state current; (ii) means forestimating a magnitude of the steady state current; and (iii) means forobtaining from the change in current with time and the magnitude of thesteady state current, a value indicative of the concentration of thereduced form or the oxidized form of the redox species.

In another aspect of the third embodiment, the cell further includes asocket region having a first contact area in electrical communicationwith the working electrode and a second contact area in electricalcommunication with the counter or counter/reference electrode, wherebythe cell may be electrically connected with at least one of the meansfor applying an electric potential difference between the electrodes andthe means for electrochemically determining the concentration of thereduced form or the oxidized form of the redox species in the liquidsample.

In a further aspect of the third embodiment, at least one of the meansfor applying an electric potential difference between the electrodes andthe means for electrochemically determining the concentration of thereduced form or the oxidized form of the redox species in the liquidsample includes a tongue plug.

In yet another aspect of the third embodiment, at least one of the meansfor applying an electric potential difference between the electrodes andthe means for electrochemically determining the concentration of thereduced form or the oxidized form of the redox species in the liquidsample includes an automated instrument for detecting an electricalsignal from the electrochemical cell and relating the electrical signalto the concentration of the reduced form or the oxidized form of theredox species in the liquid sample.

In a further aspect of the third embodiment, the cell includes asubstantially flat strip having a thickness, the strip having at leasttwo lateral edges, and wherein a notch extends through a wall of theelectrochemical cell and through the entire thickness of the strip in atleast one of the lateral edges thereof, whereby the liquid sample may beintroduced into the cell.

In still other aspects of the third embodiment, the liquid sampleincludes blood, and the redox species includes glucose.

In a fourth embodiment of the present invention, a method fordetermining a concentration of a reduced form or an oxidized form of aredox species in a liquid sample is provided, the method including: (a)providing a hollow electrochemical cell having a working electrode and acounter or counter/reference electrode wherein the working electrode isspaced from the counter or counter/reference electrode by less than 500μm; (b) applying an electric potential difference between theelectrodes; and (c) electrochemically determining the concentration ofthe reduced form or the oxidized form of the redox species in the liquidsample.

In one aspect of the fourth embodiment, step (c) includes: (i)determining a change in current with time after application of theelectric potential difference and prior to achievement of a steady statecurrent; (ii) estimating a magnitude of the steady state current; and(iii) obtaining from the change in current with time and the magnitudeof the steady state current, a value indicative of the concentration ofthe reduced form or the oxidized form of the redox species.

In another aspect of the fourth embodiment, the cell further includes asocket region having a first contact area in electrical communicationwith the working electrode and a second contact area in electricalcommunication with the counter or counter/reference electrode.

In a further aspect of the fourth embodiment, step (b) further includesthe step of: providing an automated instrument for applying an electricpotential difference between the electrodes.

In yet another aspect of the fourth embodiment, step (c) includes thesteps of: (i) providing an automated instrument for detecting anelectrical signal from the electrochemical cell; and (ii) relating theelectrical signal to the concentration of the reduced form or theoxidized form of the redox species in the liquid sample.

In a further aspect of the fourth embodiment, the cell includes asubstantially flat strip having a thickness, the strip having at leasttwo lateral edges, and wherein a notch extends through a wall of theelectrochemical cell and through the entire thickness of the strip in atleast one of the lateral edges thereof, whereby the liquid sample may beintroduced into the cell.

In still other aspects of the fourth embodiment, the liquid sampleincludes blood and the redox species includes glucose.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be particularly described by way of example onlywith reference to the accompanying schematic drawings wherein:

FIG. 1 shows the product of manufacturing step 2 in plan.

FIG. 2 shows the product of FIG. 1 in side elevation.

FIG. 3 shows the product of FIG. 1 in end elevation.

FIG. 4 shows the product of manufacturing step 3 in plan.

FIG. 5 shows the product of FIG. 4 in cross-section on line 5-5 of FIG.4.

FIG. 6 shows the product of manufacturing step 5 in plan.

FIG. 7 shows the product of FIG. 6 in side elevation.

FIG. 8 shows the product of FIG. 6 in end elevation.

FIG. 9 shows the product of manufacturing step 7 in plan.

FIG. 10 is a cross-section of FIG. 9 on line 10-10.

FIG. 11 shows the product of FIG. 9 in end elevation.

FIG. 12 shows a cell according to the invention in plan.

FIG. 13 shows the call of FIG. 12 in side elevation.

FIG. 14 shows the cell of FIG. 12 in end elevation.

FIG. 15 shows a scrap portion of a second embodiment of the invention inenlarged section.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The construction of a thin layer electrochemical cell will now bedescribed by G way of example of the improved method of manufacture. C

Step 1: A sheet 1 of Melinex® (a chemically inert, and electricallyresistive Polyethylene Terephthalate [“PET”]) approximately 13 cm×30 cmand 100 micron thick was laid flat on a sheet of release paper 2 andcoated using a Number 2 MYAR bar to a thickness of 12 microns wet(approximately 2-5 microns dry) with a water-based heat activatedadhesive 3 (ICI Novacoat system using catalyst:adhesive). The water wasthen evaporated by means of a hot air dryer leaving a contact adhesivesurface. The sheet was then turned over on a release paper and thereverse side was similarly coated with the same adhesive 4, dried, and aprotective release paper 5 applied to the exposed adhesive surface. Theedges were trimmed to obtain a sheet uniformly coated on both sides withtacky contact adhesive protected by release paper.

Step 2: The sheet with protective release papers was cut into strips 7,each about 18 mm×210 mm (FIGS. 1-3).

Step 3: A strip 7 of adhesive-coated PET from step 2 with release paper2, 5 on respective sides, was placed in a die assembly (not shown) andclamped. The die assembly was adapted to punch the strip with a locatinghole 10 at each end and with for example 37 circular holes 11 each of3.4 mm diameter at 5 mm centres equi-spaced along a line betweenlocating holes 10. The area of each hole 11 is approximately 9 squaremm.

Step 4: A sheet 12 of Mylar® PET approximately 21 cm square and 135microns thick was placed in a sputter coating chamber for palladiumcoating 13. The sputter coating took place under a vacuum of between 4and 6 millibars and in an atmosphere of argon gas. Palladium was coatedon the PET to a thickness of 100-1000 angstroms. There is thus formed asheet 14 having a palladium sputter coating 13.

Step 5: The palladium coated PET sheet 14 from Step 4 was then cut intostrips 14 and 15 and a die was used to punch two location holes 16 ineach strip, at one end (FIGS. 6, 7 and 8). Strips 14 and 15 differ onlyin dimension strips 14 being 25 mm×210 mm and strips 15 being 23 mm×210mm.

Step 6: A spacer strip 7 prepared as in step 3 was then placed in a jig(not shown) having two locating pins (one corresponding to each locatinghole 10 of strip 7) and the upper release paper 2 was removed. A strip14 of palladium coated PET prepared as in step 5 was then laid over theadhesive layer, palladium surface downwards, using the jig pins to alignthe locating holes 16 with the underlying PET strip 7. This combinationwas then passed through a laminator comprising a set of pinch rollers,one of which was adapted to heat the side bearing a palladium coated PETstrip 14. The roller on the opposite side of the strip 7 was cooled. Bythis means, only the adhesive between the palladium of strip 14 and PETstrip 7 was activated.

Step 7: PET strip 7 was then turned over and located in the jig with therelease coating uppermost. The release coating was peeled off and secondpalladium coated strip 15 was placed palladium side down on the exposedadhesive surface using the locating pins to align the strips. thisassembly was now passed again through the laminator of step 6, this timewith the hot roll adjacent the palladium coated Mylar® added in step 7so as to activate the intervening adhesive (FIGS. 9, 10 and 11).

Step 8: The assembly from step 7 was returned to the die assembly andnotches 17 punched in locations so as to extend between the circularholes 11 previously punched in the Melinex® PET and the strip edge 17.Notches 16 extend so as to intercept the circumference of each circularcell. The strip was then guillotined to give 37 individual “sensorstrips”, each strip being about 5 mm wide and each having one thin layercavity cell (FIGS. 12, 13 and 14).

There is thus produced a cell as shown in FIGS. 12, 13 or 14. The cellcomprises a first electrode consisting of PET layer 12, a palladiumlayer 13, an adhesive layer 3, a PET sheet 1, a second adhesive layer 4,a second electrode comprising palladium layer 13, and a PET layer 12.Sheet 1 defines a cylindrical cell 11 having a thickness in the cellaxial direction corresponding to the thickness of the Melinex® PBETsheet layer 1 together with the thickness of adhesive layers 3 and 4.The cell has circular palladium end walls. Access to the cell isprovided at the side edge of the cell where notches 16 intersect cell11.

In preferred embodiments of the invention, a sample to be analysed isintroduced to the cell by capillary action. The sample is placed oncontact with notch 16 and is spontaneously drawn by capillary actioninto the cell, displaced air from the cell venting from the oppositenotch 16. A surfactant may be included in the capillary space to assistin drawing in the sample.

The sensors are provided with connection means for example edgeconnectors whereby the sensors may be placed into a measuring circuit.In a preferred embodiment this is achieved by making spacer 1 shorterthan palladium supporting sheets 14, 15 and by making one sheet 15 ofshorter length than the other 14. This forms a socket region 20 havingcontact areas 21, 22 electrically connected with the working and counterelectrodes respectively. A simple tongue plug having correspondingengaging conduct surfaces can then be used for electrical connection.Connectors of other form may be devised.

Chemicals for use in the cell may be supported on the cell electrodes orwalls, may be supported on an independent support contained within thecell or may be self-supporting.

In one embodiment, chemicals for use in the cell are printed onto thepalladium surface of the electrode immediately after step 1 at whichstage the freshly-deposited palladium is more hydrophilic. For example,a solution containing 0.2 molar potassium ferricyanide and 1% by weightof glucose oxidase dehydrogenase may be printed on to the palladiumsurface. Desirably, the chemicals are printed only in the areas whichwill form a wall of the cell and for preference the chemicals areprinted on the surface by means of an ink jet printer. In this manner,the deposition of chemicals may be precisely controlled. If desired,chemicals which are desirably separated until required for use may beprinted respectively on the first and second electrodes. For example, aGOD/ferrocyanide composition can be printed on one electrode and abuffer on the other. Although it is highly preferred to apply thechemicals to the electrodes prior to assembly into a cell, chemicals mayalso be introduced into the cell as a solution after step 6 or step 8 bypipette in the traditional manner and the solvent subsequently isremoved by evaporation or drying. Chemicals need not be printed on thecell wall or the electrodes and may instead be impregnated into a gauze,membrane, non-woven fabric or the like contained within, or filling, thecavity (eg inserted in cell 11 prior to steps 6 or 7). In anotherembodiment the chemicals are formed into a porous mass which may beintroduced into the cell as a pellet or granules. Alternatively, thechemicals maybe introduced as a gel.

In a second embodiment of the invention a laminate 21 is first made froma strip 14 as obtained in step 5 adhesively sandwiched between twostrips 7 as obtained from step 3. Laminate 20 is substituted for sheet 1in step 5 and assembled with electrodes as in steps 6 and 7.

There is thus obtained a cell as shown in FIG. 15 which differs fromthat of FIGS. 9 to 11 in that the cell has an annular electrode disposedbetween the first and second electrode. This electrode can for examplebe used as a reference electrode.

It will be understood that in mass production of the cell, the parts maybe assembled as a laminate on a continuous line. For example, acontinuous sheet 1 of PET could be first punched and then adhesive couldbe applied continuously by printing on the remaining sheet. Electrodes(pre-printed with chemical solution and dried) could be fed directly asa laminate onto the adhesive coated side. Adhesive could then be appliedto the other side of the punched core sheet and then the electrode couldbe fed as a laminate onto the second side.

The adhesive could be applied as a hot melt interleaving film.Alternatively, the core sheet could first be adhesive coated and thenpunched.

By drying chemicals on each electrode prior to the gluing step theelectrode surface is protected from contamination.

Although the cell has been described with reference to Mylar® andMelinex® PET, other chemically inert and electrically resistivematerials may be utilised and other dimensions chosen. The materialsused for spacer sheet 1 and for supporting the reference and counterelectrodes may be the same or may differ one from the other. Althoughthe invention has been described with reference to palladium electrodes,other metals such as platinum, silver, gold, copper or the like may beused and silver may be reacted with a chloride to form a silver/silverchloride electrode or with other halides. The electrodes need not be ofthe same metal.

Although the use of heat activated adhesives has been described, theparts may be assembled by use of hot melt adhesives, fusible laminatesand other methods.

The dimensions of the sensor may readily be varied according torequirements.

While it is greatly preferred that the electrodes cover the cell endopenings, in other embodiments (not illustrated) the electrodes do notentirely cover the cell end openings. In that case it is desirable thatthe electrodes be in substantial overlying registration.

Preferred forms of the invention in which the electrodes cover theapertures of cell 11 have the advantages that the electrode area isprecisely defined simply by punching hole 11. Furthermore the electrodesso provided are parallel, overlying, of substantially the same area, andare substantially or entirely devoid of “edge” effects.

Although in the embodiments described each sensor has one cell cavity,sensors may be provided with two or more cavities. For example, a secondcavity may be provided with a predetermined quantity of the analyte andmay function as a reference cell.

As will be apparent to those skilled in the art from the teaching hereincontained, a feature of one embodiment herein described may be combinedwith features of other embodiments herein described or with otherembodiments described in our co-pending application. Although the sensorhas been described with reference to palladium electrodes and aGOD/ferrocyanide chemistry, it will be apparent to those skilled in theart that other chemistries, and other materials of construction may beemployed without departing from the principles herein taught.

1-32. (canceled)
 33. A biosensor for use in determining a concentrationof a component in a liquid sample, the biosensor comprising: anelectrochemical cell, the electrochemical cell comprising a firstelectrically resistive substrate having a first electrically conductivematerial on a first face, a second electrically resistive substratehaving a second electrically conductive material on a second face, thesubstrates being disposed with the first electrically conductivematerial facing the second electrically conductive material and beingseparated by at least one insulating sheet, the cell further comprisinga wall, a reagent that is deposited on at least a portion of the wall,and a sample introduction aperture formed in the insulating sheet,whereby a sample may be introduced into the cell.
 34. The biosensor ofclaim 33, wherein the electrochemical cell further comprises a workingelectrode and a counter or counter/reference electrode, wherein theworking electrode is spaced from the counter or counter/referenceelectrode by less than about 500 microns.
 35. The biosensor of claim 33,wherein the electrochemical cell further comprises a working electrodeand a counter or counter/reference electrode, wherein the workingelectrode is spaced from the counter or counter/reference electrode byless than about 200 microns.
 36. The biosensor of claim 33, wherein theaperture defines a working electrode area in the cell.
 37. The biosensorof claim 33, wherein the reagent comprises potassium ferricyanide andglucose oxidase dehydrogenase.
 38. The biosensor of claim 33, whereinthe reagent is deposited onto at least a portion of the wall by ink jetprinting.
 39. The biosensor of claim 33, wherein the reagent isdeposited onto at least one of the electrically conductive materials.40. The biosensor of claim 33, further comprising at least one adhesivelayer that is positioned between the first and second electricallyconductive materials.
 41. A biosensor for use in determining aconcentration of a component in a liquid sample, the biosensorcomprising: a thin layer electrochemical cell, the cell comprising: (a)an electrically resistive sheet comprising an aperture; (b) a firstelectrode layer covering the aperture on a first side of theelectrically resistive sheet; (c) a second electrode layer covering theaperture on a second side of the electrically resistive sheet, wherein awall of the aperture cooperates with the electrode layers to define atleast a portion of the cell wall; (d) a reagent that is deposited on atleast a portion of the cell wall; and (e) a passage for admission of aliquid sample into the aperture.
 42. The biosensor of claim 41, furthercomprising a measuring circuit.
 43. The biosensor of claim 41, whereinthe electrochemical cell further comprises a working electrode and acounter or counter/reference electrode, wherein the working electrode isspaced from the counter or counter/reference electrode by less thanabout 500 microns.
 44. The biosensor of claim 41, wherein the aperturedefines a working electrode area in the cell.
 45. The biosensor of claim41, wherein the reagent comprises about 0.2 molar potassium ferricyanideand about 1% by weight of glucose oxidase dehydrogenase.
 46. Thebiosensor of claim 41, wherein the reagent is deposited onto at least aportion of the cell wall by ink jet printing.
 47. The biosensor of claim41, wherein the reagent is deposited onto at least one of the first andsecond electrode layers.
 48. The biosensor of claim 41, wherein thefirst and second electrode layers are separated by at least oneadditional layer.
 49. The biosensor of claim 48, wherein the at leastone additional layer is an adhesive layer.